DESC:
FIELD OF THE INVENTION
The present invention provides a process for the preparation of Molnupiravir.

BACKGROUND OF THE INVENTION
Molnupiravir (compound of formula I) is an oral antiviral drug developed for the treatment of mild-to-moderate coronavirus disease (COVID-19) in adults. It is a prodrug of the synthetic nucleoside derivative N4-hydroxycytidine, and exerts its antiviral action through introduction of copying errors during viral RNA replication.

WO 2019113462 A1 discloses the five steps process for the synthesis of Molnupiravir with uridine as the starting material. The process consists of acetonide protection of secondary alcohol groups, esterification, triazole coupling, hydroxyamination and deprotection. However, there are some challenges regarding large-scale production; so there is need to develop efficient, cost effective and an industrially applicable process for the preparation of Molnupiravir.

We have developed concise and cost efficient biocatalytic route to prepare Molnupiravir from less expensive & easily available cytidine. The process involves hydroxyamination of cytosine ring followed by biocatalysed selective esterification of primary alcohol group without use of any protecting group. This method offers several significant advantages in aspects of the yield, the cost and the operations.

SUMMARY OF THE INVENTION
The present invention relates to a process for the preparation of Molnupiravir.

DETAILED DESCRIPTION OF THE INVENTION
In one aspect, the present invention provides a process for preparing Molnupiravir of formula (I)

comprising the steps of:
(a) hydroxyamination of cytidine in the presence of a suitable solvent to give the N-hydroxy cytidine of the formula (II);

(b) selective biocatalytic esterification of N-Hydroxy cytidine of formula (II) using suitable enzyme in the presence of a suitable solvent to give the Molnupiravir of formula (I).

The process according to this invention is described in detail. The reaction conditions such as solvents and temperature given are meant to provide preferred ranges and examples for the respective transformation that can be principally applied but are not supposed to restrict them to the selection given. Starting materials that may be used as the input for the process of the present invention may be obtained by any process including the process described in the art.

Hydroxyamination of cytidine can be carried out using hydroxylamine or its salts in presence of suitable solvent. Preferably, this transamination step is carried out in presence of either NH2OH.H2SO4, NH2OH.HCl or NH2OH.AcOH. The said reaction may be carried out at a temperature from about -10°C to 100°C.

The reaction of step (a) can be carried out in suitable solvent selected from the group consisting of alcohols, amides, sulphoxides, pyrrolidones, ethers, hydrocarbons, ketones, esters, nitriles, water or mixtures thereof in any suitable proportion. Examples of suitable solvent includes but not limited to methanol, ethanol, isopropanol, butanol, iso-butanol, ethyl acetate, methyl acetate, tertiarybutyl acetate, iso-propyl acetate, acetone, methylisobutyl ketone, methylethyl ketone, diethyl ketone, dimethyl ketone, toluene, ethyl ether, methyl ether, diisopropylether, methyltertbutyl ether, cyclopentylmethyl ether, dioxane, tetrahydrofuran, 2-methyltetrahydrofuran, N,N-dimethylformamide, N-methyl acetamide, N,N-dimethylacetamide, dimethylsulphoxide, N-methylpyrrolidone dichloromethane, dimethoxyethane, acetonitrile, xylene, water or mixture of these solvents in any suitable proportion.

The isolation of step (a) can be effected, if desired, by any suitable separation or purification procedure such as, for example, filtration, centrifugation, decantation, extraction, acid-base treatment, crystallization, conventional isolation and refining means such as concentration, concentration under reduced pressure, solvent extraction, crystallization, or by a combination of these procedures.

The intermediates obtained in this step may be directly used for the next step with or without isolation or it may be further purified, if isolated, to improve the purity of the product.

Esterification of N-hydroxy cytidine would need to be selective for primary alcohol group. Lipase enzymes are non-toxic, recyclable and eco-friendly biocatalysts applied in various chemical reactions. In particular Candida antarctica lipase B (CALB) is highly enantioselective and efficient biocatalyst used in the wide range of organic reactions such as esterification. Numerous studies were performed in order to improve the properties of CALB as a catalyst. One of the method is immobilization by different techniques such as non-covalent adsorption, deposition, single covalent attachment, multiple covalent attachments, entrapment in a polymeric gel in membrane or capsule, cross-linking of an enzyme and enzyme crystals. Immobilized enzymes show better catalytic efficiency than the corresponding free enzymes. Furthermore, immobilization allows ease removal of the catalyst from reaction mixture which can be reuse even up to ten times.

Fermase CALB®, a commercial Candida antarctica lipase B immobilised on polyacrylate beads may be used as a catalyst to accomplish the esterification. Fermase CALB® 1000 & Fermase CALB® 10000 shows significant thermal stability, specially under elevated temperatures.

The present invention also involves the use of Lipozyme TL IM (Source: Novozyme), Lypozyme RM 1M (source: Novozyme) and Addzyme 015 (Source: Advanced Enzyme) for the selective acetylation of N-hydroxy cytidine. Lipozyme TL IM, a commercial immobilized lipase from Thermomyces lanuginosus. Lipozyme® RM IM is a 1,3 specific lipase originating from Rhizmucor miehei which is immobilized on a resin carrier.

Immobilized CALB, Fermase CALB™, Lipozyme TL IM, Lipozyme® RM IM & Addzyme 015 Enzyme could be recycled and thereby decrease the raw material costs associated with this route.

The present invention involves the use of cheap and readily available isobutyric anhydride for the acetylation of N-hydroxy cytidine. In another aspect, acetone isobutyryl oxime ester can be used for acetylation of N-hydroxy cytidine.

The selective esterification of step (b) can be carried out in suitable solvent selected from the group consisting of alcohols, amides, sulphoxides, pyrrolidones, ethers, hydrocarbons, ketones, esters, nitriles, water or mixtures thereof in any suitable proportion. Examples of suitable solvent includes but not limited to methanol, ethanol, isopropanol, butanol, iso-butanol, ethyl acetate, methyl acetate, tertiarybutyl acetate, iso-propyl acetate, acetone, methylisobutyl ketone, methylethyl ketone, diethyl ketone, dimethyl ketone, toluene, ethyl ether, methyl ether, diisopropylether, methyltertbutyl ether, cyclopentylmethyl ether, dioxane, tetrahydrofuran, 2-methyltetrahydrofuran, N,N-dimethylformamide, N-methyl acetamide, N,N-dimethylacetamide, dimethylsulphoxide, N-methylpyrrolidone dichloromethane, dimethoxyethane, acetonitrile, xylene, water or mixture of these solvents in any suitable proportion.

The temperature at which the above steps may be carried out in between about -30°C and
about 200°C, preferably in between 0°C and l50°C, based on the solvent or mixture of solvent used in particular step.

The obtained Molnupiravir may be isolated using conventional techniques known in the art (i.e., by any suitable separation or purification procedure such as, for example, filtration, centrifugation, extraction, acid-base treatment, decantation, crystallization, conventional isolation and refining means such as concentration, concentration under reduced pressure, solvent-extraction, crystallization, or by a combination of these procedures). After separation, the solid may optionally be washed with a suitable solvent. The obtained compound may optionally be further dried. Drying may be suitably carried out in equipment such as tray dryer, vacuum oven, air oven, fluidized bed dryer, spin flash dryer, flash dryer and the like. The drying may be carried out at temperature about 40°C to about 60°C, optionally under reduced pressure. The drying may be carried out for any time periods necessary for obtaining a product with desired purity such as from about 1 hour to about 25 hours or longer.

In yet another aspect of the present invention, Molnupiravir prepared according to the processes of the present invention can be substantially pure having a chemical purity greater than about 99% by weight as determined using high performance liquid chromatography and provide overall good yield.

The compound of formula (I) preferably have enantiomeric excess of at least 95%, in particular at least 97%, more particularly at least 98%. The term "enantiomerically pure" refers to >95%, >98%, >99% and 100% enantiomeric excess (ee) of one of the enantiomers as determined by HPLC.

In yet another aspect, the present invention provides pharmaceutical composition comprising Molnupiravir prepared according to process of present invention with one or more pharmaceutically acceptable excipients. The term “pharmaceutically acceptable excipients” used in the pharmaceutical composition of invention comprise but are not limited to diluents, binders, pH stabilizing agents, disintegrants, surfactants, glidants and lubricants known in the art.

One skilled in the art will recognize that additional starting compounds and/or reagents are commercially available or may be easily prepared according to conventional methods well known to these skilled in the art.

EXAMPLES
Following Examples are set forth to aid in the understanding of the invention, and are not intended and should not be interpreted as a limitation thereon. The reaction conditions such as solvents and temperature given are meant to provide preferred ranges and examples for the respective transformation that can be principally applied but are not supposed to restrict them to the selection given. Modifications to solvents, reaction conditions, for example, temperature, duration of the reaction or combinations thereof, are envisioned as part of the present invention.

Starting materials that may be used as the input for the process of the present invention may be obtained by any process including the process described in the art. Cytidine can be obtained commercially or prepared by any method known in the art

Table: Screening Results
SN Enzyme Source Solvent HPLC Conversion
% Unreacted (NHC) % Product
1 Fermase CALB 10000/- Biocatalyst CAL BTA 10000 Fermenta Biotech 2-MeTHF 4.27% 84.02%
Acetone 5.99% 69.52%
1,4-Dioxane 3.42% 48.3%
2 Novozyme-435 Novozyme 2-MeTHF 3.3% 82.96%
Acetone 6.5% 77.3%
1,4-Dioxane 0.91% 57.97%
3 Addzyme-015 Advanced Enzyme 2-MeTHF 2.24% 73.12%
Acetone 16.38% 75.17%
1,4-Dioxane 3.57% 52.28%
4 Lypozyme TL IM Novozyme 2-MeTHF 19.66% 49.51%
Acetone 0.47% 42.36%
5 Lypozyme RM Novozyme 2-Me-THF 12.74% 57.32%

Reaction conditions: N-hydroxy cytidine monohydrate (50 mg), Enzyme (10 mg, 20 wt%), solvent (1 mL, 20 V), temp. 50 °C and reaction was stirred 17h.

Example 1
Preparation of Molnupiravir
Stage-1 Preparation of N-hydroxy cytidine Monohydrate

To a 500 mL three-neck round bottom flask equipped with an overhead stirrer and internal temperature probe was charged cytidine (100 g, 1 eq.), hydroxylamine sulphate (102 g, 1.5 eq.) and distilled water (200 mL). The mixture was heated to 70 °C and further stirred for 5 hours at the same temperature. After completion of reaction, the suspension was allowed to slowly cool to ambient temperature (25 °C) over the course of approximately 3-5 hours; then cooled to 0 -5 °C and stirred for additional 3-4 hours. The solids were isolated by vacuum filtration through Buchner funnel, washed with ice cold water and dried under vacuum oven for an overnight to afford a white crystalline solid (Yield: 80%, Weight: 90 g, HPLC purity: >95 %)

Stage-2 Preparation of Molnupiravir from N-hydroxy cytidine monohydrate

To a 250 mL three-neck round bottom flask equipped with an overhead stirrer, internal temperature probe was charged N-hydroxy cytidine monohydrate (25 g, 1.0 equiv.), isobutyric anhydride (35.7g g, 2.5 equiv.), 2-methyltetrahydrofuran (125 mL) and Fermase CALB 10000/- Biocatalyst CALB TA 10000 (3.75 g, 15 wt%) in sequence. The mixture was stirred and heated to 40 °C for 22 hours. The heating was turned off and the reaction mixture was allowed to cool to ambient temperature (25 °C). The enzyme was filtered and wash the enzyme with 2-methyltetrahydrofuran. The combined organic layer was transferred to 250 mL round bottom flask and hydroxylamine 50% in water was added. The mixture was stirred at 20 °C for 3.5 hours. The solvent was removed under reduced pressure and strip off with MTBE and again charged methyl tert-butylether, stirred at 20 °C for 5 hours. The reaction mass was filtered through Buchner funnel, washed with MTBE. The obtained solid was transferred to 100 mL round bottom flask equipped with an overhead stirrer and charged with water. The suspension was stirred with 50 RPM and heated at 70 °C to get clear solution. The solution was allowed to cool to 20 °C and stirred for 20 hours. The solids were collected by filtration through Buchner funnel, washed with ice-cold water, dried under vacuum at 50 °C for an overnight to afford a white solid product (Weight: 17.5 g; Yield: 60%; HPLC purity: 99.57%).

Example 2
Preparation of Molnupiravir
prepared according to procedure described for stage-2 of Example 1 from N-hydroxy cytidine monohydrate (25 g, 1.0 equiv.), isobutyric anhydride (35.7g g, 2.5 equiv.), 2-methyltetrahydrofuran (125 mL) and Addzyme 015 Enzyme (3.75 g) to afford a title product (Weight: 17.0 g; HPLC purity: 99.4%).
,CLAIMS:1. A process for preparing Molnupiravir of formula (I)

comprising the steps of:
(a) hydroxyamination of cytidine in the presence of a suitable solvent to give the N-hydroxy cytidine of the formula (II);

(b) selective biocatalytic esterification of N-Hydroxy cytidine of formula (II) using suitable enzyme in the presence of a suitable solvent to give the Molnupiravir of formula (I).

2. The process of claim 1, wherein in step a) hydroxyamination is carried out by using hydroxylamine or its salts.

3. The process of claim 1, wherein in step a) hydroxyamination is carried out by using NH2OH.H2SO4, NH2OH.HCl or NH2OH.AcOH.

4. The process of claim 1, wherein the acylating reagents used in step b) is isobutyric anhydride.

5. The process of claim 1, wherein the enzyme used in step b) is Lipase.

6. The process of claim 1, wherein the enzyme used in step b) is selected from the group consisting of Immobilized CALB, Fermase CALB® 1000, Fermase CALB® 10000, Lipozyme TL IM, Lipozyme® RM IM and Addzyme 015.

7. The process of claim 1, wherein the suitable solvent of step a) and step (b) is selected from the group consisting of alcohols, amides, sulphoxides, pyrrolidones, ethers, hydrocarbons, ketones, esters, nitriles, water or mixtures thereof.

8. The process of claim 1, wherein the suitable solvent of step a) and step (b) is selected from the group consisting of methanol, ethanol, isopropanol, butanol, iso-butanol, ethyl acetate, methyl acetate, tertiarybutyl acetate, iso-propyl acetate, acetone, methylisobutyl ketone, methylethyl ketone, diethyl ketone, dimethyl ketone, toluene, ethyl ether, methyl ether, diisopropylether, methyltertbutyl ether, cyclopentylmethyl ether, dioxane, tetrahydrofuran, 2-methyltetrahydrofuran, N,N-dimethylformamide, N-methyl acetamide, N,N-dimethylacetamide, dimethylsulphoxide, N-methylpyrrolidone dichloromethane, dimethoxyethane, acetonitrile, xylene, water or mixture thereof.